Between 50-80% of infants in the neonatal intensive care unit are dependent on opiates by the end of their medical treatment. Best current medical practice is to provide declining amounts of opiates to taper the infant patient off the drugs. As such, this prolongs hospitalization and constitutes a significant burden on the health care system, and an emotional burden on the family whose child remains in the hospital, and has unknown consequences for subsequent development of the newborn. Therefore opiate dependence in the neonate is a substantial clinical and public health problem in need of new therapeutic approaches. We know little about opiate dependence in the neonate, except that it differs in important mechanisms from dependence in the adult, when there is interplay between opioids and the immune system. Whether that interplay is present or not in the infant has not been studied and thus is not known, despite how common infection is in these babies. Our extensive preliminary data in a rat model show that activating the immune system during chronic morphine treatment around weaning worsens both the physical and affective aspects of withdrawal. The same immune activating treatment in very young infant rats has no effect. These behavioral changes are accompanied by age-dependent differences in the expression of immune markers in the spinal cord and brain. In this exploratory R21 application, we propose multiple analytic approaches to define the role of the immune system in morphine dependent/withdrawn infant rats. We propose careful behavioral assays that we have developed over the years to measure the behavioral and affective components of opiate withdrawal. We assess mRNA and protein for immune markers and proinflammatory cytokines in the brain and spinal cord of morphine treated infants. Our working hypothesis is that up- or down-regulating the immune system will not alter withdrawal in the young infant rat (7 days of age; human equivalent is ~full term infant), and that as the animal matures, immune system-opioid interactions begin to function in ways similar to those of the adult. Our mechanistic follow-up hypothesis is that the toll-like-receptor 4 (TLR4) mediates the interactions of opiates and the immune system to modulate opiate dependence in the older infant but has no consequence for the younger infant. To test these hypotheses, we propose pharmacological treatments and tests with mutant mice to understand the role the immune system in general and of the TLR4 receptors in particular in opiate dependent infants. The completion of this work will define how the interactions between opioid and immune systems change as the animal matures. In determining if immune modulation is effective or not in the opiate dependent infant, we will provide preclinical data to direct clinical decisions as to whether or not immune based therapeutics should be developed and implemented for the treatment of opiate dependence in infant patient.